Complex formation between NOD2 CARDab and RIP2-CARD.

Abstract

<p>(A) NOD2 CARDab with a N-terminal GST-tag and RIP2-CARD equipped with a N-terminal GB1-tag and a C-terminal His₆-tag were co-expressed and pulled-down with glutathione sepharose beads. From left: Lane 1) Protein Marker, GE Healthcare. Lane 2) Soluble lysate. Lane 3) Bead eluate after 3C-protease cleavage. NOD2-CARDab 22.0 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. Lane 4) Supernatant bound to beads. GST-NOD2 CARDab 48.5 kDa (green) and GB1-RIP2 CARD-His 18.6 kDa (red) are indicated by arrows. (B) Effect of NOD2 CARDab single point mutations on RIP2 CARD binding. A representative cross section of the mutants tested are shown. GST-NOD2 CARDab 48.5 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. * = residual expression of GST. (C) Effect of RIP2 CARD single point mutations on NOD2 CARDab binding. A cross section of the mutants tested are shown. GST-NOD2 CARDab 48.5 kDa and GB1-RIP2 CARD-His 18.6 kDa are indicated by green and red arrows, respectively. (D) Effect of RIP2 CARD single point mutations on NOD1 CARD binding. A cross section of the mutants tested are shown. GST-NOD1 CARD 40.9 kDa (aa17–138) and GB1-RIP2 CARD-His 18.6 kDa are indicated by blue and red arrows, respectively.</p